Caveolin-1 functions as a novel Cdc42 guanine nucleotide dissociation inhibitor in pancreatic beta-cells

The cycling of the small Rho family GTPase Cdc42 is required for insulin granule exocytosis, although the regulatory proteins involved in Cdc42 cycling in pancreatic beta-cells are unknown. Here we demonstrate that the caveolar protein caveolin-1 (Cav-1) is a Cdc42-binding protein in beta-cells. Cav-1 associated with Cdc42-VAMP2-bound granules present near the plasma membrane under basal conditions. However, stimulation with glucose induced the dissociation of Cav-1 from Cdc42-VAMP2 complexes, coordinate with the timing of Cdc42 activation. Analyses of the Cav-1 scaffolding domain revealed a motif conserved in guanine nucleotide dissociation inhibitors (GDIs), which suggested a novel role for Cav-1 as a Cdc42 GDI in beta-cells. The novel role was further supported by: 1) in vitro binding analyses that demonstrated a direct interaction between Cav-1 and Cdc42; 2) GST-Cdc42 interaction assays showing preferential Cav-1 binding to GDP-Cdc42 over that of GTP-Cdc42; 3) Cav-1 depletion studies resulting in an inappropriate 40% induction of activated Cdc42 in the absence of stimuli and also a 40% increase in basal insulin release from both MIN6 cells and islets. Expression of wild-type Cav-1 in Cav-1-depleted cells restored basal level secretion to normal, whereas expression of a scaffolding domain mutant of Cav-1 failed to normalize secretion. Taken together, these data suggest that Cav-1 functions as a Cdc42 GDI in beta-cells, maintaining Cdc42 in an inactive state and regulating basal secretion in the absence of stimuli. Through its interaction with the Cdc42-VAMP2-bound insulin granule complex, Cav-1 may contribute to the specific targeting of granules to "active sites" of exocytosis organized by caveolae.     

Crystallization of the c14-rotor of the chloroplast ATP synthase reveals that it contains pigments

The ATP synthase is one of the most important enzymes on earth as it couples the transmembrane electrochemical potential of protons to the synthesis of ATP from ADP and inorganic phosphate, providing the main ATP source of almost all higher life on earth. During ATP synthesis, stepwise protonation of a conserved carboxylate on each protein subunit of an oligomeric ring of 10-15 c-subunits is commonly thought to drive rotation of the rotor moiety (c(10-14)gammaepsilon) relative to stator moiety (alpha(3)beta(3)deltaab(2)). Here we report the isolation and crystallization of the c(14)-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 A. Though ATP synthase was not previously known to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revealed that they contain 1 chlorophyll and 2 carotenoids, thereby showing for the first time that the chloroplast ATP synthase contains cofactors, leading to the question of the possible roles of the functions of the pigments in the chloroplast ATP synthase.     

Efficiency of ATP synthase as a molecular machine

The experimentally measured effect of the odd magnetic isotope ²⁵Mg on the rate of ATP synthesis by the mitochondrial F₀F₁ enzyme is used to compare the rates of the main reactions in the catalytic site, in the framework of a radical-ion process. The rate-limiting step of synthesis is the addition of the ADP oxyradical to the phosphate P=O bond. The relationship of the rate constants deduced from the magnetic isotope effect shows that the mechanochemical efficiency of ATP synthase operating with spinless ²⁴Mg or ²⁶Mg nuclei will not exceed 50%. The performance is nearly doubled with magnetic ²⁵Mg.     

Zinc modulation of water permeability reveals that aquaporin 0 functions as a cooperative tetramer

We previously showed that the water permeability of AQP0, the water channel of the lens, increases with acid pH and that His40 is required (Németh-Cahalan, K.L., and J.E. Hall. 2000. J. Biol. Chem. 275:6777-6782; Németh-Cahalan, K.L., K. Kalman, and J.E. Hall. 2004. J. Gen. Physiol. 123:573-580). We have now investigated the effect of zinc (and other transition metals) on the water permeability of AQP0 expressed in Xenopus oocytes and determined the amino acid residues that facilitate zinc modulation. Zinc (1 mM) increased AQP0 water permeability by a factor of two and prevented any additional increase induced by acid pH. Zinc had no effect on water permeability of AQP1, AQP4 or MIPfun (AQP0 from killifish), or on mutants of AQP1 and MIPfun with added external histidines. Nickel, but not copper, had the same effect on AQP0 water permeability as zinc. A fit of the concentration dependence of the zinc effect to the Hill equation gives a coefficient greater than three, suggesting that binding of more than one zinc ion is necessary to enhance water permeability. His40 and His122 are necessary for zinc modulation of AQP0 water permeability, implying structural constraints for zinc binding and functional modulation. The change in water permeability was highly sensitive to a coinjected zinc-insensitive mutant and a single insensitive monomer completely abolished zinc modulation. Our results suggest a model in which positive cooperativity among subunits of the AQP0 tetramer is required for zinc modulation, implying that the tetramer is the functional unit. The results also offer the possibility of a pharmacological approach to manipulate the water permeability and transparency of the lens.     

Lipid raft proteome reveals ATP synthase complex in the cell surface

Since detergent-resistant lipid rafts are involved in pathogen invasion, cholesterol homeostasis, angiogenesis, neurodegenerative diseases and signal transduction, protein identification in the rafts could provide important information to study their function. Here, we analyzed detergent-resistant raft proteins isolated from rat liver by capillary liquid chromatography-tandem mass spectrometry. Out of 196 proteins identified, 32% belonged to the raft or plasma membrane, 24% to mitochondrial, 20% to microsomal, 7% to miscellaneous, and 17% are unknown proteins. For example, membrane-bound receptors, trimeric GTP-binding proteins, ATP-binding cassette transporters, and glycosylphosphatidylinositol-anchored proteins were identified in this analysis. Unexpectedly, there were many mitochondrial proteins, raising a new issue for the presence of mitochondrial rafts or the localization of mitochondrial proteins into plasma membrane rafts. We confirmed that ATP synthase alpha and beta were expressed on the surface of the plasma membrane in HepG2 hepatocytes by immunofluorescence, cell surface biotinylation, and cellular fractionation. They had two distinct biochemical properties, detergent insolubility and low density, suggesting that the ATP synthase complex might be located in plasma membrane rafts as well as in the mitochondria.     

The efficiency of ATP synthase as a molecular machine

On the basis of the experimentally measured isotope effect of magnesium (25)Mg on the rate of ATP synthesis by mithochondrial ATP synthase, the rate constants of the reactions in the catalytic site have been estimated. The limiting step of the synthesis is shown to be the addition of8phosphoryl anion-radical of ADP to the P=O bond of phosphate with the rate constant of 1,2 x 10(8) s(-1). From the ratio of the rate constants, the mechanochemical efficiency of ATP synthase was estimated to be about 10% for enzymes with (24, 26)Mg isotopes and to be doubled for enzymes with (25)Mg in the catalytic site.     

Evidence that an isoform of calpain-10 is a regulator of exocytosis in pancreatic beta-cells

Calpain-10 (CAPN10) is the first type 2 diabetes susceptibility gene to be identified through a genome scan, with polymorphisms being associated with altered CAPN10 expression. Functional data have been hitherto elusive, but we report here a corresponding increase between CAPN10 expression level and regulated insulin secretion. Pancreatic beta-cell secretory granule exocytosis is mediated by the soluble N-ethylmaleimide-sensitive fusion protein attachment receptor protein complex of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin 1, and vesicle-associated membrane protein 2. We report, for the first time, direct binding of a calpain-10 isoform with members of this complex. Furthermore, SNAP-25 undergoes a Ca2+-dependent partial proteolysis during exocytosis, with calpain protease inhibitor similarly suppressing both insulin secretion and SNAP-25 proteolysis. Based upon these findings, we postulate that an isoform of calpain-10 is a Ca2+-sensor that functions to trigger exocytosis in pancreatic beta-cells.     

Hippocalcin functions as a calcium sensor in hippocampal LTD

It is not fully understood how NMDAR-dependent LTD causes Ca(2+)-dependent endocytosis of AMPARs. Here we show that the neuronal Ca(2+) sensor hippocalcin binds the beta2-adaptin subunit of the AP2 adaptor complex and that along with GluR2 these coimmunoprecipitate in a Ca(2+)-sensitive manner. Infusion of a truncated mutant of hippocalcin (HIP(2-72)) that lacks the Ca(2+) binding domains prevents synaptically evoked LTD but has no effect on LTP. These data indicate that the AP2-hippocalcin complex acts as a Ca(2+) sensor that couples NMDAR-dependent activation to regulated endocytosis of AMPARs during LTD.     

Adenine nucleotide regulation in pancreatic beta-cells: modeling of ATP/ADP-Ca2+ interactions

Glucose metabolism stimulates insulin secretion in pancreatic beta-cells. A consequence of metabolism is an increase in the ratio of ATP to ADP ([ATP]/[ADP]) that contributes to depolarization of the plasma membrane via inhibition of ATP-sensitive K+ (K(ATP)) channels. The subsequent activation of calcium channels and increased intracellular calcium leads to insulin exocytosis. Here we evaluate new data and review the literature on nucleotide pool regulation to determine the utility and predictive value of a new mathematical model of ion and metabolic flux regulation in beta-cells. The model relates glucose consumption, nucleotide pool concentration, respiration, Ca2+ flux, and K(ATP) channel activity. The results support the hypothesis that beta-cells maintain a relatively high [ATP]/[ADP] value even in low glucose and that dramatically decreased free ADP with only modestly increased ATP follows from glucose metabolism. We suggest that the mechanism in beta-cells that leads to this result can simply involve keeping the total adenine nucleotide concentration unchanged during a glucose elevation if a high [ATP]/[ADP] ratio exits even at low glucose levels. Furthermore, modeling shows that independent glucose-induced oscillations of intracellular calcium can lead to slow oscillations in nucleotide concentrations, further predicting an influence of calcium flux on other metabolic oscillations. The results demonstrate the utility of comprehensive mathematical modeling in understanding the ramifications of potential defects in beta-cell function in diabetes.     

Cytokine-induced inhibition of insulin release from mouse pancreatic beta-cells deficient in inducible nitric oxide synthase

Cytokines may participate in islet destruction during the development of type 1 diabetes. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1 beta or (IL-1 beta + IFN-gamma) may impair islet function in rodent islets. Inhibition of iNOS or a deletion of the iNOS gene (iNOS -/- mice) protects against cytokine-induced beta-cell suppression, although cytokines might also induce NO-independent impairment. Presently, we exposed wild-type (wt, C57BL/6 x 129SvEv) and iNOS -/- islets to IL-1 beta (25 U/ml) and (IL-1 beta (25 U/ml) + IFN-gamma (1000 U/ml)) for 48 h. IL-1 beta and (IL-1 beta + IFN-gamma) induced a significant increase in NO formation in wt but not in iNOS -/- islets. Both IL-1 beta and (IL-1 beta + IFN-gamma) impaired glucose-stimulated insulin release and reduced the insulin content of wt islets, while (IL-1 beta + IFN-gamma) reduced glucose oxidation rates and cell viability. IL-1 beta exposure to iNOS -/- islets impaired glucose-stimulated insulin release, increased insulin accumulation and reduced the insulin content, without any increase in cell death. Exposure to (IL-1 beta + IFN-gamma) had no effect on iNOS -/- islets except reducing the insulin content. Our data suggest that IL-1 beta may inhibit glucose-stimulated insulin release by pathways that are not NO-dependent and not related to glucose metabolism or cell death.     

Vascular abnormalities in mice deficient for the G protein-coupled receptor GPR4 that functions as a pH sensor

GPR4 is a G protein-coupled receptor expressed in the vasculature, lung, kidney, and other tissues. In vitro ectopic overexpression studies implicated GPR4 in sensing extracellular pH changes leading to cyclic AMP (cAMP) production. To investigate its biological roles in vivo, we generated GPR4-deficient mice by homologous recombination. Whereas GPR4-null adult mice appeared phenotypically normal, neonates showed a higher frequency of perinatal mortality. The average litter size from GPR4(-/-) intercrosses was approximately 30% smaller than that from GPR4(+/+) intercrosses on N3 and N5 C57BL/6 genetic backgrounds. A fraction of knockout embryos and neonates had spontaneous hemorrhages, dilated and tortuous subcutaneous blood vessels, and defective vascular smooth muscle cell coverage. Mesangial cells in kidney glomeruli were also significantly reduced in GPR4-null neonates. Some neonates exhibited respiratory distress with airway lining cell metaplasia. To examine whether GPR4 is functionally involved in vascular pH sensing, an ex vivo aortic ring assay was used under defined pH conditions. Compared to wild-type aortas, microvessel outgrowth from GPR4-null aortas was less inhibited by acidic extracellular pH. Treatment with an analog of cAMP, a downstream effector of GPR4, abolished microvessel outgrowth bypassing the GPR4-knockout phenotype. These results suggest that GPR4 deficiency leads to partially penetrant vascular abnormalities during development and that this receptor functions in blood vessel pH sensing.     

Batten disease fibroblasts in culture accumulate mitochondrial ATP synthase subunit 9.

Batten Disease is a lysosomal storage disease in which the major component that accumulates is subunit 9 of mitochondrial ATP synthase. Whether or not fibroblasts in culture exhibit this phenotype is controversial. We show that fibroblasts from a human Batten Disease patient and from a mouse model of this disease exhibit autofluorescent inclusion bodies. We also demonstrate that levels of ATP synthase subunit 9 are elevated in these diseased fibroblasts when compared to control cells. However, the exact growth state of the human fibroblasts was critical, and this factor probably accounts for discrepencies in the literature.     

Neurotensin is a regulator of insulin secretion in pancreatic beta-cells

Neurotensin (NT) is secreted from neurons and gastrointestinal endocrine cells. We previously reported that the three NT receptors (NTSRs) are expressed in pancreatic islets and beta cell lines on which we observed a protective effect of NT against cytotoxic agents. In this study, we explored the role of NT on insulin secretion in the endocrine pancreatic beta cells. We observed that NT stimulates insulin secretion at low glucose level and has a small inhibiting effect on stimulated insulin secretion from isolated islets or INS-1E cells. We studied the mechanisms by which NT elicited calcium concentration changes using fura-2 loaded islets or INS-1E cells. NT increases calcium influx through the opening of cationic channels. Similar calcium influxes were observed after treatment with NTSR selective ligands. NT-evoked calcium regulation involves PKC and the translocation of PKCα and PKC? to the plasma membrane. Part of NT effects appears to be also mediated by PKA but not via the Erk pathway. Taken together, these data provide evidence for an important endocrine role of NT in the regulation of the secretory function of beta cells.     

Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene

Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome. To enable analysis of this mutation in control nuclear backgrounds, two different cell lines were transformed with mitochondria carrying NARP mutant mitochondrial DNA. Transformant cell lines had decreased ATP synthesis capacity, and many also had abnormally high levels of two ATP synthase sub-complexes, one of which was F(1)-ATPase. A combination of metabolic labeling and immunoblotting experiments indicated that assembly of ATP synthase was slowed and that the assembled holoenzyme was unstable in cells carrying NARP mutant mitochondrial DNA compared with control cells. These findings indicate that altered assembly and stability of ATP synthase are underlying molecular defects associated with the NARP mutation in subunit 6 of ATP synthase, yet intrinsic enzyme activity is also compromised.     

Inhibition of electrical activity in mouse pancreatic beta-cells by the ATP/ADP translocase inhibitor, bongkrekic acid

Bongkrekic acid causes fatal food poisoning which is associated with hyperglycaemia. Here we demonstrate that bongkrekic acid, a potent inhibitor of the mitochondrial ATP/ADP translocase, inhibits glucose-induced electrical activity in the pancreatic beta-cell through the stimulation of ATP-sensitive potassium channel (K-ATP-channel) activity. By comparison of its effects with those of oligomycin, we suggest that bongkrekic acid acts by the inhibition of glucose metabolism and may induce hyperglycaemia by impairing beta-cell function.